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Assays of matrix metalloproteinases (MMPs) activities: A review

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DOI: 10.1016/j.biochi.2005.01.007 · Source: PubMed

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 Biochimie 87 (2005) 265–272
www.elsevier.com/locate/biochi

Assays of matrix metalloproteinases (MMPs) activities: a review

Carine Lombard, Joëlle Saulnier, Jean Wallach *
Laboratoire de biochimie analytique et synthèse bioorganique, UFR Chimie-Biochimie, Université Claude-Bernard Lyon 1,
69622 Villeurbanne cedex, France

Received 26 October 2004; accepted 13 January 2005

Abstract

Measurement of matrix metalloproteinase (MMP) activity often remains a challenge, mainly in complex media. Two sets of methods are
currently used. The first one measures the hydrolysis of natural protein substrates (labeled or not) and includes the popular zymography. These
techniques which are quite sensitive, cannot generally be carried out on a continuous basis. The second one takes mainly advantage of the
increase of fluorescence, which is associated to the hydrolysis of initially quenched fluorogenic peptide substrates. Quite recently, another
group, which is a compromise between the other two, has been developed. It measures the hydrolysis of synthetic triple-helical peptide
substrates. These different methods are described and discussed.
© 2005 Elsevier SAS. All rights reserved.

Keywords: Metalloprotease assay; Fluorescent substrates; Immunocapture; Triple-helical peptides

1. Introduction not always figuring the biological activity of the enzymes,

The choice of an assay for measuring the activity of an
enzyme is critically depending on the qualitative and quanti-
tative objectives of the study. This is particularly important in
the case of the matrix metalloproteinases (MMPs), as their
natural substrates are usually insoluble (or poorly soluble)
proteins, complex mixtures of proteins and associate macro-
molecules, structural components of extracellular matrices.
They are synthesized as inactive zymogens and must be enzy-
matically activated for being active. Furthermore, their activi-
ties are in vivo regulated by endogenous inhibitors (TIMPs).
For measuring their enzymatic activity, the choice is between
easy-to-use techniques, generally with synthetic substrates,
Abbreviations: A2pr, 2,3-diaminopropionic acid; Adp, 2-amino-3-(6,7-
dimethoxy-4-coumaryl)propionic acid; APMA, p-aminophenylmercuric ace-
tate; Ctmr, 5-carboxytetramethylrhodamine; DNFB, dinitrofluorobenzene;
Dnp, dinitrophenyl; Dpa, diphenylalanine; DTNB, dithiobis-(2-nitrobenzoic
acid); FIA, flow-injection analysis; FITC, fluorescein isothiocyanate; HTS,
high-throughput screening; HRP, horseradish peroxidase; Mca, 7-methoxy-
coumarin-2-acetic acid; MOCAc, (7-methoxycoumarin-4-yl)acetyl; Nma,
N-methylanthranilic acid; TNBSA, 2,4,6-trinitrobenzene sulfuric acid; TNF,
tumor necrosis factor; uPA, urokinase-type plasminogen activator.
* Corresponding author.
E-mail address: jean.wallach@univ-lyon1.fr (J. Wallach).
0300-9084/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.biochi.2005.01.007
and more cumbersome and complex methods using native or
modified proteins which are more difficult to use for screen-
ing. In the case of studies aimed at determining the role of
MMPs, it is obviously better to use native proteins or deriva-
tives as substrates whereas the screening of inhibitors, in pres-
ence of purified enzymes, will be easier with synthetic sub-
strates, which allow to perform rapid and numerous assays
within quite a short time and high-throughput screening. The
situation becomes more complicated when assays are carried
out in biological fluids, due to the presence of interfering mol-
ecules, mainly inhibitors and also due to the fact that MMP
specificities are similar.
In many circumstances, immunochemical procedures can
replace enzymatic assays, but they cannot discriminate
between zymogens and active enzymes.
The different assays of matrix metalloproteinase activities
may be roughly classified in:
• Assays using proteins (modified or not);
• Assays using synthetic linear peptides and their deriva-
tives;
• Assays using synthetic “mini-collagens”.
In this brief review, we will describe, illustrate, and compare
these assays, referring to recent published results from vari-
ous laboratories.
266 C. Lombard et al. / Biochimie 87 (2005) 265–272
2. Assays using proteins
Ideally, it would be better to measure the MMP activities
with their most specific native proteins as substrates. But the
hydrolysis rates are low and the classical methods are not
sensitive enough. This is the reason why current methods pref-
erably use labeled proteins, whereas some other original pro-
cedures have also been described.
2.1. Non-modified proteins
While collagenolytic activity may be evaluated by the spec-
trophotometric assay of hydroxyproline in the hydrolysates,
this technique is more valuable for bacterial metalloprotein-
ases than for matrix ones [1]. Contrastingly, cleavage rates of
insoluble and soluble collagens by tissue collagenases could
be measured from dansylation at the N-terminal residues of
the cleaved peptides, after hydrolysis. Extensive hydrolysis
and HPLC separation were monitored by fluorescence mea-
surements. Using such a technique, kinetic parameters could
be determined (KM values were calculated to be in the micro-
molar range) [2]. A similar approach has been conducted by
Mecham et al., in order to characterize the cleavage sites in
insoluble elastin. Prior to Edman sequencing of hydrolyzed
peptides, they used DNFB, which reacts covalently with pri-
mary and secondary amino groups. They could then identify
′ Zymography has also been applied to in situ studies. Ini-
the P1 residues of proteolytic hydrolysis. Theoretically, it
would be possible to quantify the cleavage rates, even if it
would obviously be a tedious method [3].
Very recently, three collagenase assays (MMP-1, MMP-8,
and MMP-13) with triple-helical collagens as substrates, have
been developed using capillary gel electrophoresis with laser-
induced dynamic fluorescence detection, with a non-covalent
fluorescent dye (NanoOrange). A good linearity of peak areas
was obtained over each assay range (15–150 ng/tube for
MMP-1, 3-30 ng/tube for MMP-8, and 1.5–30 ng/tube for
MMP-13). The method has been applied to the estimation of
inhibitors and the results well agree with those obtained with
fluorescent peptides [4].
A great number of data concerning MMP activities has
been obtained by zymography, which allows the visualiza-
tion of protease activities. This technique involves the elec-
trophoresis of enzymes through polyacrylamide gels contain-
ing copolymerized protease substrate. It has been used for
many years mainly with gelatin as a substrate. Electrophore-
sis is generally performed in sodium dodecylsulfate (SDS)
and proteases are then renatured, enabling the hydrolysis of
the substrate. After staining, the zones of digestion may be
evidenced and/or quantified. In the early 90s, zymography
was used to analyze the gelatinolytic activities of secreted
proteinases of metastatic cell lines [5–7], but it was only in
1994 that the technique became quantitative. With the help of
a purified MMP-2/TIMP-2 complex, Kleiner and Stetler-
Stevenson could detect picogram quantities of the enzyme. A
linear relationship could be evidenced between 10 and 120 pg
after a 18 h-digestion period. After a 43 h-incubation, the
detection limit was lowered to 2 pg. Initial rates of hydrolysis
could be determined from density measurements within 30 h
of digestion for enzyme concentrations ranging from 0.1 to
4 ng MMP-2 [8]. In a similar way, but using a single-step
staining method, which led to fast and reproducible results,
Leber and Balkwill [9] could demonstrate a detection limit
of 32 pg for pro-MMP-9 and a linear range below 1 ng. More
recently, Gogly et al. reported a detection limit as low as 0.1 pg
in the case of an MMP-1 assay on collagen zymograms. Such
a sensitivity is comparable to an immunodot blot assay using
chemiluminescence but it may be noticed that immunoas-
says do not discriminate between pro- and active forms of
enzyme which is not the case for zymography, in the experi-
mental conditions described by the authors. The specificity
of this method for collagenase was demonstrated, as no lysis
could be observed with 300 pg of either MMP-2 or MMP-3
[10].
Unlike gelatinases, matrilysin (MMP-7), and collagena-
ses (MMP-1 and MMP-13) are difficult to detect. In a recent
report, it was demonstrated that heparin may enhance the
assays, lowering the detection limit to 30 pg for MMP-7 and
to 0.2 ng for MMP-1 and MMP-13. The mechanism of activ-
ity enhancement is not fully explained, but it has been sug-
gested that heparin may act by inducing a conformation
change, facilitating refolding, reducing autolysis or helping
anchoring of the enzyme to the gel [11].
tially, limited to soft and homogenous tissues, it has recently
been used to demonstrate the sub-lamellar location of gelati-
nases in hard epidermal layers and a comparison has been
made with an adjacent soft connective tissue matrix. The
authors could then evaluate the influence of tissue topogra-
phy on the technique [12].
Zymography may also be performed in a reverse mode,
designed to detect the presence of metalloproteinase inhibi-
tors. In this case, a proteinase is directly incorporated into the
substrate-containing gel. The method was applied for the
quantitative assessment of metalloproteinase inhibitor activ-
ity either with gelatin [13] or FITC-labeled collagen and
casein [14] as substrates.
Today, zymography is probably one of the most popular
techniques for measuring MMP activities. Some experimen-
tal procedures have recently been detailed and discussed in
the case of enzymatic activities in a crude medium of cul-
tured synoviocytes [15].
2.2. Labeled proteins
Various methods have been developed, using chemically
modified or engineered proteins. In the first category, the most
popular are proteins labeled either by radioelements or by
fluorescent groups, but recent results have emphasized the
usefulness of other modifications.
2.2.1. Radiolabeled proteins
For sensitivity and ease of detection, labeling by means of
[3H] acetic anhydride is frequently performed. Assays have
 C. Lombard et al. / Biochimie 87 (2005) 265–272 267

been developed with casein [16], collagens (native or dena- TNBSA, due to the liberation of new reactive amino groups.
tured) [17] and elastin [3]. It is also possible to use [125I] Applied to MMP-2 and -9, the assay is quite rapid (<1 h),
gelatin [18]. In any case, undegraded proteins are precipi- specific and may be used for the evaluation of inhibition [25].
tated with trichloracetic acid and radioactivity counted in the This methodology has recently been applied to measure the
supernatant. inhibition of MMP-1, -2 and -9 by means of pycnogenol
metabolites [26].
2.2.2. Fluorescently labeled proteins
Fluorescein and other derivatives have been used for a long 2.2.5. Immunocapture assay
time as protein modifiers, allowing sensitive fluorometric A new general principle for measurement of proteolytic
assays. Nevertheless, all these assays require a precipitation activities was developed in 1997 by Verheijen et al. [27]. Sum-
step, keeping them as endpoint assays. For a continuous moni- marized in Fig. 1, it is based on the activation of pro-
urokinase in urokinase and further spectrophotometric assay,
toring of the reaction, it has recently been proposed to use
using a specific chromogenic peptide substrate. The method
dyes, which are linked in such a way that fluorescence is
may be extended to metalloproteinase assay by replacing the
quenched. Protease-catalyzed hydrolysis relieves this quench-
activation site present in pro-urokinase-type plasminogen acti-
ing, yielding a dramatic increase in fluorescence [19]. Deriva-
vator (pro-uPA), namely Pro-Arg-Phe-Lys-Ile-Ile-Gly-Gly, by
tives of elastin, collagen, and gelatin are now commercially
a sequence recognized by MMPs. A comparison between pri-
available (Molecular Probes and others) and enzymatic activ-
mary sequences of the recognition site of various MMPs led
ity may be monitored with fluorescence microplate readers
the authors to choose the introduction of the sequence: Arg-
or spectrofluorometers.
Pro-Leu-Gly-Ile-Ile-Gly-Gly in a mutant pro-uPA, obtained
Flow cytometric analysis is another method for measuring
with the recombinant circle PCR method. A coupled assay
the MMP activity with fluorescent substrates, such as fluo-
was then developed with pyroGlu-Gly-Arg-pNA as a sub-
rescein isothiocyanate (FITC)-labeled gelatin, after coating
strate for modified urokinase. The parabolic curve absor-
on polystyrene microspheres [20]. This technique has recently
bance vs. time could be transformed into a linear assay by
been employed as a routine method to measure MMP-2 and
plotting absorbance changes against square time. The sensi-
MMP-9 activities and MMP-9 inhibition by means of a pep-
tivity was the highest for MMP-2 and MMP-9, intermediate
tidomimetic based on the consensus sequence of the cleav-
for MMP-1 and much lower for MMP-3 and MMP-7. The
age sites in type II collagen [21].
assay could be made specific for MMP-9 [28] and MMP-2
[29] using specific monoclonal antibodies. By means of these
2.2.3. Biotinylated gelatin
antibodies, MMPs were captured from complex media and
A simple, inexpensive, and highly sensitive assay of
activities specifically analysed. Simple modifications could
MMP-2 (and, at a lower extent, of MMP-9), using biotiny-
raise the sensitivity to 60 pg ml–1 for MMP-9 activity assay
lated gelatin, was reported in 2000 by Ratnikov et al. [22].
[30]. This method, now commercially available (Amersham
After a proteolytic hydrolysis, steptavidin-coated plates were
BioSciences) has been further successfully used for activity
used to capture the hydrolyzed biotin-containing fragments.
measurements and inhibitor assessments [31,32].
Activity was measured with a streptavidin-peroxidase (HRP)
complex added to the medium. As two biotin residues are
necessary for binding both streptavidin and streptavidin- 3. Assays using linear peptides
HRP, the hydrolysis leads to cleavage products devoid of
biotin or possessing only one biotin and a decrease in absor- The first substrates used for measuring peptidasic MMPs
bance is observed. This 2 h-assay allowed a measurement of activities were linear peptides corresponding to the scissible
the gelatinolytic MMP-2 activity for concentrations as low as
0.16 ng ml–1. Recently, with minor modifications, this tech-
nique was applied to the demonstration of the activation of
pro-MMP-9 by APMA or trypsin. The authors could obtain a
linear relationship of the absorbance decrease vs. the enzyme
concentration within the range 0.15–2.5 ng ml–1 [23].

2.2.4. Succinylated gelatin

This chemical modification has been proposed in order to
develop a spectrophotometric assay, based on the measure-
ment of primary amines exposed after gelatinase hydrolysis.
Previously, applied to elastin hydrolysis by elastase [24], sub-
strate succinylation is performed with succinic anhydride,
blocking free amino groups, which cannot then react with
TNBSA. When succinylated gelatin is enzymatically hydro- Fig. 1. Colorimetric assay of matrix metalloproteinase activity using modi-
lyzed, absorbance at 450 nm increases after the action of fied pro-urokinase.
268 C. Lombard et al. / Biochimie 87 (2005) 265–272
Gly-Ile region of collagen and N-terminally labeled by a Dnp
group, such as Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg. In
this case, the Dnp-Pro-Gln-Gly peptide was either extracted
into ethylacetate and then spectrophotometrically assayed at
365 nm [33] or separated by HPLC from the reaction mixture
and estimated from its peak area after calibration [34]. Other
similar peptides were suggested including the Gly-Ile bond,
such as Dnp-Pro-Leu-Gly-Ile-Ala-Gly for MMP-8 and Dnp-
Pro-Gln-Gly-Ile-Ala for MMP-9 [35]. Weingarten et al. pro-
posed a series of peptide derivatives, including thiopeptolide,
which has a thioester bond instead of the cleavage site pep-
tide bond. After hydrolysis, the liberated thiol reacts with 5,5′-
dithiobis-(2-nitrobenzoic acid) (DTNB), forming a colored
2-nitro-5-thiobenzoic acid, which can be detected at 412 nm
[36,37]. A colorimetric assay kit based on this reaction is com-
mercially available for MMP-3 assay and kits for MMP-1,
MMP-7 and MMP-12 were developed (BioMol).
Nevertheless, most of MMPs assays using peptide sub-
strates are fluorometric and based on resonance energy trans-
fer quenching. Such an effect occurs between a fluorescent
donor group and a quenching acceptor, with an optimal dis-
tance between them. The hydrolysis of the substrate and the
separation of both fragments abolish the energy transfer and
lead to an increase of fluorescence. This general principle has
been applied for fluorimetric assays of many proteolytic
enzymes [38].
Six different fluorophores have been used in MMP fluo-
rogenic peptide substrates, together with various quenchers.
The first was tryptophan (Trp) with Dnp as the quenching
group. Stack and Gray [39] demonstrated that the presence
of Trp in P’2 position and of Dnp in P3 position leads to an
efficient substrate for both continuous collagenase and gelati-
nase assays, with kcat/KM values of 5.4 and 440 µM–1 h–1,
respectively. The optimization of the peptide sequence allowed
Netzel-Arnett et al. [40] to develop fluorescent assays for five
MMPs. Dnp has also been associated with two other fluoro-
phores, namely (7-methoxycoumarin-4-yl)acetyl (Mca) [41]
and N-methylanthranilic acid (Nma) [42], these two groups
being much more fluorescent than tryptophan. In particular,
the substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, was
extensively used for monitoring MMP activities and for
screening inhibitors. In 2004, Neumann et al. [43] improved
the properties of this substrate by N-terminal addition of a
Lys, increasing both the specificity constant and water solu-
bility.
Another very popular donor/acceptor pair is EDANS:
5-[(2-aminoethyl)amino] naphthalene-1-sulfonic acid and
DABCYL: 4-(4-dimethylaminophenylazo)benzoic acid) (a
sulfonyl derivative (DABSYL) may also be used as a
quencher). This couple has many advantages: (1) there is a
good spectral overlap between the EDANS emission spec-
trum and the DABCYL absorption spectrum, (2) EDANS has
a high fluorescence, (3) the high wavelengths limit interfer-
ence with amino acid side chain fluorescence. First intro-
duced in a substrate for assaying HIV-1 protease [44], MMP
fluorogenic substrates including these groups have also been
synthesized, EDANS being introduced either on the side chain
of Glu or at the C-terminal end of the peptides [45–47].
Two other couples have been introduced: Lucifer
Yellow/Ctmr and more recently two CyDye fluors. In the first
case, the enzyme-specific peptide sequence is placed between
a N-terminal Ser and C-terminal Lys. Serine is further oxi-
dized by periodate and coupled with Lucifer Yellow, forming
a hydrazone. C-terminal Lys is then coupled with 5-carboxy-
tetramethylrhodamine, quenching the dye fluorescence
[48,49]. Despite its interesting properties, this couple has not
been extensively used for MMP assays.
More recently, CyDye fluors were investigated for the study
of enzyme activity and the development of high-throughput
screening (HTS). The pair Cy3/Cy5Q was validated for MMP
activity assays and a comparison made with the Mca/Dnp
pair [50]. The system could be applied to a high-throughput
screening of MMP-3 inhibitors, with an imaging platform,
which gave improved reading accuracy and faster plate pro-
cessing times compared with a commercially available
microtitre plate reader [51].
A recent review summarized the state of the art on the fluo-
rogenic substrates, which has yet been described [52].
Together with data on kinetic parameters, this paper is full of
practical information concerning the synthesis of substrates,
their characterization and experimental procedures for assays.
Fluorometric assays are very convenient for MMP assays
and for screening inhibitors. Nevertheless, it has been noticed
that fluorescence changes are not linear with substrate con-
centration, due to “inner filter effects”. It has then been sug-
gested that apparent substrate inhibition observed by several
research groups should be reconsidered. This phenomenon
has been described in particular with EDANS/DABCYL in
the case of cytomegalovirus protease [53] and trypsin [54]
assays. Such an effect may influence the determination of
Michaelis–Menten parameters, if the calibration curves of
fluorescence changes are not made vs. substrate concentra-
tion. The analysis of this effect on the activity vs. concentra-
tion was done by Geoghegan [49] in the case of the TNF
convertase.
Although generally developed in cuvettes or microplates,
MMP activity may also be measured with fluorescent sub-
strate peptides by flow injection analysis (FIA). Itoh et al.
[55] have proposed a simple and convenient method for mea-
suring MMP-7 activity. Using MOCAc-Pro-Leu-Gly-Leu-
A2pr (DNP)-Ala-Arg-NH2 as a substrate, fluorescence was
measured after a 2 min-hydrolysis, leading to a linear stan-
dard curve from 5 to 100 pmol of enzyme.
4. Assays using triple-helical peptides
As mentioned earlier in this review, the study of the pre-
cise mechanism of action of MMPs and the evaluation of
inhibitors need the use of molecules which correspond, as
closely as possible, to the native substrates of the enzymes. It
is the reason why well-defined triple helical peptides, derived
 C. Lombard et al. / Biochimie 87 (2005) 265–272
Fig. 2. Schematic structure of branched triple-helical peptides.
from collagen sequences, were designed, mainly by Greg
Fields and his group. The challenge was to synthesize struc-
tures, which would be stable enough to remain in triple-
helical conformation in the conditions of assays.
Different approaches have been described. A solid-phase
synthesis was first developed, featuring a C-terminal cova-
lent branch and incorporating specific sequences between this
branch and a N-terminal (Gly-Pro-Hyp)n domain (Fig. 2)
[56,57].
Another approach was the design of “peptide-amphiphile”
triple helices, based on the non-covalent self-assembly of lipo-
philic molecules, N-terminally-linked to a peptide sequence
[58–60]. Both the covalent branch and “peptide-amphiphile”
approaches have been used for studying susceptibility of
triple-helical peptides to MMP hydrolysis and localization of
enzyme cleavage sites, with Edman degradation sequence
analysis and MALDI-MS [61,62].
In order to develop a continuous assay method, Lauer-
Fields et al. [63] designed and synthesized triple-helical pep-
tides containing fluorophores. Fluorescence quenching used
the Mca/Dnp pair, Lys(Dnp) and Lys(Mca) introduced in the
positions P′5 and P5, respectively.
Another type of triple-helical structure was designed, based
on a disulfide bridging approach. It contains a collagenase
cleavage site linked to a cystine-knot and is N-terminally
extended by (Gly-Pro-Hyp)5 triplets [64]. The addition of a
Gly-Pro-Hyp repeat near the cystine knot leads to a remark-
ably stable triple-helical structure, with a melting tempera-
ture of 41 °C [65]. As previously, done by Fields and his team,
these substrates were made fluorogenic by addition of a dan-
syl group and tryptophan, on the N-terminal position of the
a1 and a′1 chain, respectively [66]. In any case, these triple-
helical peptide substrates were hydrolyzed in a similar man-
ner to native collagens, cleavage occurring between Gly and
Ile (or Leu) according to the chain type. The kinetic param-
eters for hydrolysing several of them by various MMPs have
recently been reviewed [67,68]. In the last 2 years, different
improvements were made. As far as selectivity is concerned,
two triple-helical peptides were designed as MMP-2 and
MMP-9 selective substrates to evaluate the active MMP pro-
duction in cellular systems [69].
At the same time, studies were carried out to limit the
increase of hydrophobicity due to the introduction of the Mca
269
derivative as fluorophore. 2-Amino-3-(6,7-dimethoxy-4-
coumaryl)propionic acid (Adp), associated with Dnp as a
quencher, definitely assumed to be compatible with multi-
well plate readers and better tolerated by MMPs than
Lys(Mca) [70].
Even if these substrates are not commercially available and
difficult to synthesize, they are essential to model the MMP
activity. Very recently, the importance of their thermal stabil-
ity on the activities of different MMP has been emphasized
and may help demonstrate subtle differences in their abilities
to hydrolyze triple helices [71]. Furthermore, the study indi-
cating that collagenase unwinds triple-helical collagen prior
to peptide bond hydrolysis, clearly supports the usefulness of
triple-helical peptides as MMPs substrates to understand the
mechanism of action of MMPs [72].
5. Concluding remarks
One of the challenges of the MMP assays is the develop-
ment of sensitive, selective, and rapid methods.
If we consider the above-mentioned methodologies, none
of them completely fulfil these requirements. The use of natu-
ral substrates mimics the true MMP activities but is generally
tedious and slow. The use of fluorescent substrates is sensi-
tive, rapid but does not always figure the in vivo activity of
the enzymes. The use of triple-helical peptides seems to be a
good compromise between them, but these substrates are not
easy to synthesize and, as far as we know, not commercially
available yet.
Observations have been done concerning the role, in the
interactions proteins-MMPs, of residues distant from the pri-
mary recognition site. These relevant interactions are not
present when synthetic substrates are used [73]. Moreover,
when using fluorogenic substrates, Beekman et al. [74] could
measure MMP-13 activity, even when MMP-13 was in com-
plex with a2-macroglobulin. Such an activity could not be
evidenced with collagen as a substrate.
Direct comparison of fluorometric and zymographic meth-
ods led Quesada et al. [75] to conclude that gelatin zymogra-
phy is more sensitive than fluorometric methods using either
FITC-casein or fluorogenic peptides, but insisted on prob-
270 C. Lombard et al. / Biochimie 87 (2005) 265–272
lems associated with quantitation and they recommended the
use of fluorogenic substrates for high throughput screening
of MMP inhibition.
A recent paper is suggesting another possible orientation
for improving MMP assays, particularly in complex mix-
tures. Rasmussen et al. developed a new technique for mea-
suring activity of a mixture of MMPs. Named MEMRAS
(“Multiple-Enzyme/Multiple-Reagent Assay System”), this
technique uses substrates with different selectivity profiles
against a group of MMPs. If they solve simultaneous equa-
tions, the authors could estimate individual activities in a
MMP mixture from independent initial rate experiments [76].
Another promising axis is the future development of
protease-specific protein chips and protease-activity chips, as
suggested by Lopez-Otin and Overall [77]. With the help of
combinatorial peptide libraries, the improvement of sub-
strate selectivity will favour such an approach.
Acknowledgments
We thank Mrs C. Beaufrere for English improvement.
References
[1] J.L. Hao, T. Nagano, M. Nakamura, N. Kumagai, H. Mishima,
T. Nishida, Effect of galardin on collagen degradation by Pseudomo-
nas aeruginosa, Exp. Eye Res. 69 (1999) 595–601.
[2] S. Takahashi, S. Han, The rates of cleavage of insoluble and soluble
collagens by tissue collagenases, Connect. Tissue Res. 30 (1993)
99–116.
[3] R.P. Mecham, T.J. Broekelmann, C.J. Fliszar, S.D. Shapiro, H.G. Wel-
gus, R.M. Senior, Elastin degradation by matrix metalloproteinases.
Cleavage site specificity and mechanism of elastolysis, J. Biol. Chem.
272 (1997) 18071–18076.
[4] M. Sano, I. Nishino, K. Ueno, H. Kamimori, Assay of collagenase
activity for native triple-helical collagen using capillary gel electro-
phoresis with laser-induced fluorescence detection, J. Chromatogr. B
809 (2004) 251–256.
[5] F. Umenishi, H. Yasumitsu, Y. Ashida, J. Yamauti, M. Umeda,
K. Miyazaki, Purification and properties of extracellular matrix-
degrading metalloproteinase overproduced by Rous sarcoma virus-
transformed rat liver cell line, and its identification as transin, J.
Biochem. (Tokyo) 108 (1990) 537–543.
[6] K. Miyazaki, Y. Hattori, F. Umenishi, H. Yasumitsu, M. Umeda,
Purification and characterization of extracellular matrix-degrading
metalloproteinase, matrin (pump-1), secreted from human rectal car-
cinoma cell line, Cancer Res. 50 (1990) 7758–7764.
[7] Y. Kato,Y. Nakayama, M. Umeda, K. Miyazaki, Induction of 103-kDa
gelatinase/type IV collagenase by acidic culture conditions in mouse
metastatic melanoma cell lines, J. Biol. Chem. 267 (1992) 11424–
11430.
[8] D.E. Kleiner, W.G. Stetler-Stevenson, Quantitative zymography:
detection of picogram quantities of gelatinases, Anal. Biochem. 218
(1994) 325–329.
[9] T.M. Leber, F.R. Balkwill, Zymography: a single-step staining
method for quantitation of proteolytic activity on substrate gels, Anal.
Biochem. 249 (1997) 24–28.
[10] B. Gogly, N. Groult, W. Hornebeck, G. Godeau, B. Pellat, Collagen
zymography as a sensitive and specific technique for the determina-
tion of subpicogram levels of interstitial collagenase, Anal. Biochem.
255 (1998) 211–216.
[11] W.H. Yu, J.F. Woessner Jr., Heparin-enhanced zymographic detection
of matrilysin and collagenases, Anal. Biochem. 293 (2001) 38–42.
[12] B.A. Mungall, C.C. Pollitt, In situ zymography: topographical consid-
erations, J. Biochem. Biophys. Methods 47 (2001) 169–176.
[13] G.W. Oliver, J.D. Leferson, W.G. Stetler-Stevenson, D.E. Kleiner,
Quantitative reverse zymography: analysis of picogram amounts of
metalloproteinase inhibitors using gelatinase A and B reverse zymo-
grams, Anal. Biochem. 244 (1997) 161–166.
[14] S. Hattori, H. Fujisaki, T. Kiriyama, T. Yokoyama, S. Irie, Real-time
zymography and reverse zymography: a method for detecting activi-
ties of matrix metalloproteinases and their inhibitors using FITC-
labeled collagen and casein as substrates, Anal. Biochem. 301 (2002)
27–34.
[15] L. Troeberg, H. Nagase, Measurement of matrix metalloproteinase
activities in the medium of cultured synoviocytes using zymography,
Methods Mol. Biol. 225 (2003) 77–87.
[16] D.H. Manicourt, V. Lefebvre, An assay for matrix metalloproteinases
and other proteases acting on proteoglycans, casein, or gelatin, Anal.
Biochem. 215 (1993) 171–179.
[17] J.B. Catterall, T.E. Cawston, Assays of matrix metalloproteinases
(MMPs) and MMP inhibitors: bioassays and immunoassays appli-
cable to cell culture medium, serum and synovial fluid, Methods Mol.
Biol. 225 (2003) 353–364.
[18] T. Sorsa, T. Salo, E. Koivunen, J. Tyynelä, Y.T. Konttinen, U. Berg-
mann, et al., Activation of type IV procollagenases by human tumor-
associated trypsin-2, J. Biol. Chem. 272 (1997) 21067–21074.
[19] D.A. Menges, D.L. Ternullo, A.L. Tan-Wilson, S. Gal, Continuous
assay of proteases using a microtiter plate fluorescence reader, Anal.
Biochem. 254 (1997) 144–147.
[20] Y. St-Pierre, M. Desrosiers, P. Tremblay, P.-O. Esteve, G. Opdenakker,
Flow cytometric analysis of gelatinase B (MMP-9) activity using
immobilized fluorescent substrate on microspheres, Cytometry 25
(1996) 374–380.
[21] J. Hu, P.E. Van den Steen, M. Houde, T.T. Ilenchuk, G. Opdenakker,
Inhibitors of gelatinase B/matrix metalloproteinase-9 activity. Com-
parison of a peptidomimetic and polyhistidine with single-chain
derivatives of a neutralizing monoclonal antibody, Biochem. Pharma-
col. 67 (2004) 1001–1009.
[22] B. Ratnikov, E. Deryugina, J. Leng, G. Marchenko, D. Dembrow,
A. Strongin, Determination of matrix metalloproteinase activity using
biotinylated gelatin, Anal. Biochem. 286 (2000) 149–155.
[23] H.A. Bawadi, T.M. Antunes, F. Shih, J.N. Losso, In vitro inhibition of
the activation of pro-matrix metalloproteinase 1 (Pro-MMP-1) and
pro-matrix metalloproteinase 9 (pro-MMP-9) by rice and soybean
Bowman-Birk inhibitors, J. Agric. Food Chem. 52 (2004) 4730–4736.
[24] S.K. Rao, M. Mathrubutham, A. Karteron, K. Sorensen, J.R. Cohen, A
versatile microassay for elastase using succinylated elastin, Anal.
Biochem. 250 (1997) 222–227.
[25] V.M. Baragi, B.J. Shaw, R.R. Renkiewicz, P.J. Kuipers, H.G. Welgus,
M. Mathrubutham, et al., A versatile assay for gelatinases using
succinylated gelatin, Matrix Biol. 19 (2000) 267–273.
[26] T. Grimm, A. Schäfer, P. Högger, Antioxidant activity and inhibition
of matrix metalloproteinases by metabolites of maritime pine bark
extract (pycnogenol), Free Radic. Biol. Med. 36 (2004) 811–822.
[27] J.H. Verheijen, N.M.E. Nieuwenbroek, B. Beekman, R. Hanemaaijer,
H.W. Verspaget, H.K. Ronday, et al., Modified proenzymes as artifi-
cial substrates for proteolytic enzymes: colorimetric assay of bacterial
collagenase and matrix metalloproteinase activity using modified
pro-urokinase, Biochem. J. 323 (1997) 603–609.
[28] R. Hanemaaijer, H. Visser,Y.T. Konttinen, P. Koolwijk, J.H. Verheijen,
A novel and simple immunocapture assay for determination of gelati-
nase B (MMP-9) activities in biological fluids: saliva from patients
with Sjögren’s syndrome contain increased latent and active
gelatinase-B levels, Matrix Biol. 17 (1998) 657–665.
[29] S.J. Capper, J. Verheijen, L. Smith, M. Sully, H. Visser, R. Hane-
maaijer, Determination of gelatinase A (MMP-2) activity using a
novel immunocapture assay, Ann. N. Y. Acad. Sci. 878 (1999) 487–
490.
 C. Lombard et al. / Biochimie 87 (2005) 265–272
[30] R. Hanemaaijer, N. Van Lent, H. Visser, J. Verheijen, Methods to
increase the sensitivity of the MMP-9 activity assay, Life Science
News 3 (1999) 8–9.
[31] M. Crul, L.V. Beerepoot, E. Stokvis, J.S.P. Vermaat, H. Rosing,
J.H. Beijnen, et al., Clinical pharmacokinetics, pharmacodynamics
and metabolism of the novel matrix metalloproteinase inhibitor ABT-
518, Cancer Chemother. Pharmacol. 50 (2002) 473–478.
[32] M.A. Huisman, A. Timmer, M. Zeinstra, E.K. Serlier, R. Hanemaaijer,
H. Van Goor, et al., Matrix-metalloproteinase activity in first trimester
placental bed biopsies in further complicated and uncomplicated
pregnancies, Placenta 25 (2004) 253–258.
[33] Y. Masui, T. Takemoto, S. Sakakibara, H. Hori, Y. Nagai, Synthetic
substrates for vertebrate collagenase, Biochem. Med. 17 (1977) 215–
221.
[34] R.D. Gray, H.H. Saneii, Characterization of vertebrate collagenase
activity by high-performance liquid chromatography using a synthetic
substrate, Anal. Biochem. 120 (1982) 339–346.
[35] H.R. Williams, T.Y. Lin, Human polymorphonuclear leukocyte colla-
genase and gelatinase. Comparison of certain enzymatic properties,
Int. J. Biochem. 16 (1984) 1321–1329.
[36] H. Weingarten, R. Martin, J. Feder, Synthetic substrates of vertebrate
collagenase, Biochemistry 24 (1985) 6730–6734.
[37] H. Weingarten, J. Feder, Spectrophotometric assay for vertebrate
collagenase, Anal. Biochem. 147 (1985) 437–440.
[38] C.G. Knight, Fluorimetric assays of proteolytic enzymes, Methods
Enzymol. 248 (1995) 18–34.
[39] M.S. Stack, R.D. Gray, Comparison of vertebrate collagenase and
gelatinase using a new fluorogenic substrate peptide, J. Biol. Chem.
264 (1989) 4277–4281.
[40] S. Netzel-Arnett, S.K. Mallya, H. Nagase, H. Birkedal-Hansen,
H.E. Van Wart, Continuously recording fluorescent assays optimized
for five human matrix metalloproteinases, Anal. Biochem. 195 (1991)
86–92.
[41] C.G. Knight, F. Willenbrock, G. Murphy, A novel coumarin-labelled
peptide for sensitive continuous assays of matrix metalloproteinases,
FEBS Lett. 296 (1992) 263–266.
[42] D.M. Bickett, M.D. Green, J. Berman, M. Dezube, A.S. Howe,
P.J. Brown, et al., A high throughput fluorogenic substrate for intersti-
tial collagenase (MMP-1) and gelatinase (MMP-9), Anal. Biochem.
212 (1993) 58–64.
[43] U. Neumann, H. Kubota, K. Frei, V. Ganu, D. Leppert, Characteriza-
tion of Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, a fluorogenic
substrate with increased specificity constants for collagenases and
tumor necrosis factor converting enzyme, Anal. Biochem. 328 (2004)
166–173.
[44] E.D. Matayoshi, G.T. Wang, G.A. Krafft, J. Erickson, Novel fluoro-
genic substrates for assaying retroviral proteases by resonance energy
transfer, Science 247 (1990) 954–958.
[45] J.W. Drijfhout, J. Nagel, B. Beekman, J.M. Te Koppele, W. Bloem-
hoff, Solid phase synthesis of peptides containing the fluorescence
energy transfer Dabcyl–Edans couple, in: P.T.P. Kaumaya,
R.S. Hodges (Eds.), “Peptides: Chemistry, Structure and Biology”,
1996, pp. 129–131 pp.
[46] B. Beekman, J.W. Drijfhout, W. Bloemhoff, H.K. Ronday, P.P. Tak,
J.M. Te Koppele, Convenient fluorometric assay for matrix metallo-
proteinase activity and its application in biological media, FEBS Lett.
390 (1996) 221–225.
[47] B. Beekman, B. van El, J.W. Drijfhout, H.K. Ronday, J.M. Te Kop-
pele, Highly increased levels of active stromelysin in rheumatoid
synovial fluid determined by a selective fluorogenic assay, FEBS Lett.
418 (1997) 305–309.
[48] K.F. Geoghegan, M.J. Emery, W.H. Martin, A.S. McColl,
G.O. Daumy, Site-directed double fluorescent tagging of human renin
and collagenase (MMP-1) substrate peptides using the periodate oxi-
dation of N-terminal serine. An apparently general strategy for provi-
sion of energy-transfer substrates for proteases, Bioconjug. Chem. 4
(1993) 537–544.
271
[49] K.F. Geoghegan, Improved method for converting an unmodified
peptide to an energy-transfer substrate for a proteinase, Bioconjug.
Chem. 7 (1996) 385–391.
[50] J. Peppard, Q. Pham, A. Clark, D. Farley, Y. Sakane, R. Graves, et al.,
Development of an assay suitable for high-throughput screening to
measure matrix metalloprotease activity, Assay Drug Dev. Technol. 1
(2003) 425–433.
[51] J. George, M.L. Teear, C.G. Norey, D.D. Burns, Evaluation of an
imaging platform during the development of a FRET protease assay, J.
Biomol. Screen. 8 (2003) 72–80.
[52] G.B. Fields, Using fluorogenic peptide substrates to assay matrix
metalloproteinases, Methods Mol. Biol. 151 (2001) 495–518.
[53] B.P. Holskin, M. Bukhtiyarova, B.M. Dunn, P. Baur, J. de Chastonay,
M.W. Pennington, A continuous fluorescence-based assay of human
cytomegalovirus protease using a peptide substrate, Anal. Biochem.
226 (1995) 148–155.
[54] S. Grahn, D. Ullmann, H.-D. Jakubke, Design and synthesis of fluo-
rogenic trypsin peptide substrates based on resonance energy transfer,
Anal. Biochem. 265 (1998) 225–231.
[55] M. Itoh, M. Osaki, T. Chiba, K. Masuda, T. Akizawa, M. Yoshioka,
et al., Flow injection analysis for measurement of activity of matrix
metalloproteinase-7 (MMP-7), J. Pharm. Biomed. Anal. 15 (1997)
1417–1426.
[56] C.G. Fields, C.M. Lovdahl, A.J. Miles, V.L. Mathias-Hagen,
G.B. Fields, Solid-phase synthesis and stability of triple-helical pep-
tides incorporating native collagen sequences, Biopolymers 33 (1993)
1695–1707.
[57] C.G. Fields, D.J. Mickelson, S.L. Drake, J.B. McCarthy, G.B. Fields,
Melanoma cell adhesion and spreading activities of a synthetic 124-
residue triple-helical ‘mini-collagen’, J. Biol. Chem. 268 (1993)
14153–14160.
[58] Y.-C. Yu, P. Berndt, M. Tirrell, G.B. Fields, Self-assembling
amphiphiles for construction of protein molecular architecture, J. Am.
Chem. Soc. 118 (1996) 12515–12520.
[59] Y.-C. Yu, M. Tirrell, G.B. Fields, Minimal lipidation stabilizes
protein-like molecular architecture, J. Am. Chem. Soc. 120 (1998)
9979–9987.
[60] Y.-C. Yu, V. Roontga, V.A. Daragan, K.H. Mayo, M. Tirrell,
G.B. Fields, Structure and dynamics of peptide amphiphiles incorpo-
rating triple-helical protein like molecular architecture, Biochemistry
38 (1999) 1659–1668.
[61] J.L. Lauer-Fields, H. Nagase, G.B. Fields, Use of Edman degradation
sequence analysis and matrix-assisted laser desorption/ionization
mass spectrometry in designing substrates for matrix metalloprotein-
ases, J. Chromatogr. A 890 (2000) 117–125.
[62] J.L. Lauer-Fields, K.A. Tuzinski, K. Shimokawa, H. Nagase,
G.B. Fields, Hydrolysis of triple-helical collagen peptide models by
matrix metalloproteinases, J. Biol. Chem. 275 (2000) 13282–13290.
[63] J.L. Lauer-Fields, T. Broder, T. Sritharan, L. Chung, H. Nagase,
G.B. Fields, Kinetic analysis of matrix metalloproteinase activity
using fluorogenic triple-helical substrates, Biochemistry 40 (2001)
5795–5803.
[64] J. Ottl, R. Battistuta, M. Pieper, H. Tschesche, W. Bode, K. Kühn,
et al., Design and synthesis of heterotrimeric collagen peptides with a
built-in cystine-knot. Models for collagen catabolism by matrix-
metalloproteases, FEBS Lett. 398 (1996) 31–36.
[65] J. Ottl, L. Moroder, Disulfide-bridged heterotrimeric collagen pep-
tides containing the collagenase cleavage site of collagen type I.
Synthesis and conformational properties, J. Am. Chem. Soc. 121
(1999) 653–661.
[66] J.C.D. Müller, J. Ottl, L. Moroder, Heterotrimeric collagen peptides as
fluorogenic collagenase substrates: synthesis, conformational proper-
ties, and enzymatic digestion, Biochemistry 39 (2000) 5111–5116.
[67] J.L. Lauer-Fields, D. Juska, G.B. Fields, Matrix metalloproteinases
and collagen catabolism, Biopolymers 66 (2002) 19–32.
[68] J.L. Lauer-Fields, G.B. Fields, Triple-helical peptide analysis of col-
lagenolytic protease activity, Biol. Chem. 383 (2002) 1095–1105.
272 C. Lombard et al. / Biochimie 87 (2005) 265–272
[69] J.L. Lauer-Fields, T. Sritharan, M.S. Stack, H. Nagase, G.B. Fields,
Selective hydrolysis of triple-helical substrates by matrix metallopro-
teinase-2 and -9, J. Biol. Chem. 278 (2003) 18140–18145.
[70] J.L. Lauer-Fields, P. Kele, G. Sui, H. Nagase, R.M. Leblanc,
G.B. Fields, Analysis of matrix metalloproteinase triple-helical pepti-
dase activity with substrates incorporating fluorogenic L- or D- amino
acids, Anal. Biochem. 321 (2003) 105–115.
[71] D. Minond, J.L. Lauer-Fields, H. Nagase, G.B. Fields, Matrix metal-
loproteinase triple-helical peptidase activities are differentially regu-
lated by substrate stability, Biochemistry 43 (2004) 11474–11481.
[72] L. Chung, D. Dinakarpandian, N. Yoshida, J.L. Lauer-Fields,
G.B. Fields, R. Visse, et al., Collagenase unwinds triple-helical col-
lagen prior to peptide bond hydrolysis, EMBO J. 23 (2004) 3020–
3030.
View publication stats
[73] S. Marini, G.F. Fasciglione, G. de Sanctis, S. D’Alessio, V. Politi,
M. Coletta, Cleavage of bovine collagen I by neutrophil collagenase
MMP-8. Effect of pH on the catalytic properties as compared to
synthetic substrates, J. Biol. Chem. 275 (2000) 18657–18663.
[74] B. Beekman, J.W. Drijfhout, H.K. Ronday, J.M. Te Koppele, Fluoro-
genic MMP activity assay for plasma including MMPs complexed to
alpha 2-macroglobulin, Ann. N. Y. Acad. Sci. 878 (1999) 150–158.
[75] A.R. Quesada, M.M. Barbacid, E. Mira, P. Fernandez-Resa, G. Mar-
quez, M. Aracil, Evaluation of fluorometric and zymographic methods
as activity assays for stromelysins and gelatinases, Clin. Exp.
Metastasis 15 (1997) 26–32.
[76] F.H. Rasmussen, N. Yeung, L. Kiefer, G. Murphy, C. Lopez-Otin,
M.P. Vitek, et al., Use of a multiple-enzyme/multiple-reagent assay
system to quantify activity levels in samples containing mixtures of
matrix metalloproteinases, Biochemistry 43 (2004) 2987–2995.
[77] C. Lopez-Otin, C.M. Overall, Protease degradomics: a new challenge
for proteomics, Nat. Rev. Mol. Cell Biol. 3 (2002) 509–519.